1. ** Synaptic fraction H P1 P2 S2 PSD95 CREB
Sophie Laguesse date experiment number SL45
Aim: Test Prosapip1 expression after synaptic fractionation from Nac of mice after binge drinking
Method: Mice experienced intermittent access 20% alcohol 2 bottle choice drinking session during eight weeks. Control animals had access only to water. 30 minutes after the beginning of alcohol session, rats were sacrificed and Nac were immediately removed.
Nac were homogenated in 250uL cold Krebs buffer+sucrose 0,32M+phosphatase/protease inhibitors using a needle. 80ul were kept as total homogenate (H) and were lysed by adding 80uL RIPA buffer. The samples were centrifuged 10min at g and the supernatant was removed (=S1). 250 ul of Krebs-glucose were added to the pellet and samples were centrifuged a second time at 1000g 10min 4deg. Supernatant was removed and added to S1 (=S1 total). 180ul RIPA buffer was added to the pellet (=P1). S1 total samples were centrifuged 20min 16000g 4deg. Supernatant was kept for quality control (=S2) and 180uL RIPA buffer was added to the pellet (=P2). All P2 fractions were centrifuged 10min 10000g and supernatant were used for protein concentration determination. Prosapip1 and Actin expression were determined by Western Blot.
Quality control of synaptic fractionation: PSD95 and CREB expression were determined by WB for one control (C3) and one binge sample (B3).
Synaptic fraction
Prosapip1 in synaptic fraction
Water
Alcohol (binge) 50
100
150
200 **
Prosapip1/actin (% of the control)
Water
Alcohol (binge)
Prosapip1
Actin
Quality control B3 sample
H P1 P2 S2
PSD95
CREB
Conclusion: Prosapip1 expression is significantly increased in the Nac of mice after binge drinking in the isolated crude synaptic fraction. This confirm the increased expression observed in the total homogenate of Nac and confirm that Prosapip1 is present at the synapses.

2. Sophie Laguesse date 2015.07.10 experiment number SL50
Aim: Test Prosapip1 expression after synaptic fractionation from motor Cortex of mice after binge drinking
Method: Mice experienced intermittent access 20% alcohol 2 bottle choice drinking session during eight weeks. Control animals had access only to water. 30 minutes after the beginning of alcohol session, rats were sacrificed and Nac were immediately removed.
Nac were homogenated in 250uL cold Krebs buffer+sucrose 0,32M+phosphatase/protease inhibitors using a needle. 80ul were kept as total homogenate (H) and were lysed by adding 80uL RIPA buffer. The samples were centrifuged 10min at g and the supernatant was removed (=S1). 250 ul of Krebs-glucose were added to the pellet and samples were centrifuged a second time at 1000g 10min 4deg. Supernatant was removed and added to S1 (=S1 total). 180ul RIPA buffer was added to the pellet (=P1). S1 total samples were centrifuged 20min 16000g 4deg. Supernatant was kept for quality control (=S2) and 180uL RIPA buffer was added to the pellet (=P2). All P2 fractions were centrifuged 10min 10000g and supernatant were used for protein concentration determination. Prosapip1 and Actin expression were determined by Western Blot.
Synaptic fraction
Prosapip1 in synaptic fraction
Water
Alcohol (binge) ns
100 80
Prosapip1
Prosapip1/actin
(% of the control) 60
Actin 40 20
water
alcohol (binge)
Conclusion: As expected, Prosapip1 expression is not increased in the motor cortex of mice after binge drinking in the isolated crude synaptic fraction.